Project Summary The intestine is the highest turnover tissue in the body, making it an excellent model of adult stem cell biology. Constant renewal makes the gut susceptible to colorectal cancer and injury, such as radiation toxicity. Intestinal stem cells (ISCs) are integral to the renewal of the intestine and hence to many of its numerous pathologies. Despite the clinical significance of ISCs, two unreconciled models of their identity and behavior exist. The hierarchical model argues that two distinct ISC types exist ? rare quiescent stem cells (QSCs) and more abundant, rapidly-dividing crypt base columnar stem cells (CBCs). Both CBCs and QSCs self-renew and differentiate, but QSCs can only come from other QSCs. On the other hand, the continuum model argues that CBCs are the only true stem cells and that they can exist in a continuum of states, including quiescence. Extensive efforts to resolve these two models have been unsuccessful and contradictory. The primary source of confusion has been an overreliance on purported cell type-specific promoters, which mark overlapping and heterogeneous cell populations. A novel, unbiased, and precision approach is required to probe the existence of a distinct QSC population. With this in mind, we will combine phylogenetic inference with CRISPR/Cas9 genome editing to thoroughly map the intestinal cellular division tree without the use of biased promoters. We will generate transgenic mice that contain the elements of the CRISPR/Cas9 system, as well as a short synthetic DNA barcode sequence that is targeted by Cas9. As intestinal cells divide, their barcodes are cut and repaired by the mutation-prone pathway of nonhomologous end-joining (NHEJ), producing an enormous diversity of heritable mutations. Barcodes are then sequenced from single cells, along with the transcriptome, providing a complete picture of both cellular lineage history and identity. By applying this system to the intestine, we hypothesize that we will observe lineage trees matching the predictions of the hierarchical model, namely a continuous QSC lineage that all other cells branch from. The existence of a continuous QSC lineage would act to maintain genome integrity in the face of replicative and metabolic stress, which carries implications for carcinogenesis and aging biology. More broadly, our approach will serve as a template for probing tissue development, maintenance, injury, and neoplasia across a variety of tissues with unprecedented depth.